unraveling survival strategies
DESCRIPTION
Unraveling Survival Strategies. Valina L. Dawson, Ph.D. Johns Hopkins Institute for Cell Engineering. Too Many Cell Types in the Nervous System?. Cell Type Estimates:. Outside the Brain ~300 Nervous System ~1,500-5,000. Two types of cell death?! (apoptosis or necrosis). - PowerPoint PPT PresentationTRANSCRIPT
Unraveling Survival Strategies
Valina L. Dawson, Ph.D.Johns Hopkins Institute for Cell
Engineering
Too Many Cell Types in the Nervous System?
Cell Type Estimates:
Outside the Brain ~300
Nervous System ~1,500-5,000
C.F. Stevens Curr. Biol. 8:708
Two types of cell death?!
(apoptosis or necrosis)
Ordering the Cell Death PathwayCauseTrigger
Contributor Consequence
Cell Death
Identification of Novel Neuronal Survival Pathways
Strategies
Preconditioning
TRENDS in Neurosciences 26(5): 248-254, 2003
Preconditioning is a phenomenon in which “any stimulus capable of causing injury to a tissue or organ can, when applied close to (but below) the threshold of damage, activate endogenous protective mechanisms, thus potentially lessening the impact of subsequent, more severe stimuli.”
Preconditioning and TolerancePreconditioning Stimuli: ischemia, oxygen glucose deprivation, excitotoxins, reactive oxygen species, cytokines/ceramide, activation of adenosine receptors or KATP channels, spreading depression, inhibition of oxidative phosphorylation.
Acute Tolerance: Occurs immediately following preconditioning, is sustained for several hours and most often associated with post-translational protein modifications.
Sustained Tolerance: Maintained for several days and requires new protein synthesis
Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain?
CONCLUSIONS: The beneficial effect of TIAs on lesion size in ADC and T2 suggests the existence of endogenous neuroprotection in the human brain.
Stroke 2004 Mar;35(3):616-21.2004
CONCLUSIONS: This study suggests that ischemic tolerance may play a role in patients with ipsilateral TIAs before cerebral ischemia, allowing better recovery from a subsequent ischemic stroke.
Neurology 2000 Jun 13;54(11):2089-94.
Stroke 1999 Sep;30(9):1851-4CONCLUSIONS: Assuming that a TIA represents an adequate stimulus to elicit ischemic tolerance, the results suggest that ischemic tolerance might occur in the human brain.
Ca2+
NMDA-R
Calmodulin
NOS NO
TranscriptionFactors
Nucleus
Ras-GDP
Ras-GTPRafMekErk
?
Proc. Natl. Acad. Sci, USA 95: 5773-5778 (1998)Proc. Natl. Acad. Sci., USA., 97: 436-441 (2000).
Nitric Oxide Mediates NMDA Receptor-Induced Activation of p21ras & Preconditioning.
New Protein SynthesisSurvival ProteinsExpressed
Functional Cloning StrategyPrimary Cortical Neurons - Preconditioned with 10 min OGD
Harvest mRNA
cDNA Library
Insert Library into a Retrovirus
Infect Fibroblast Cultures
Amplify viable cell clones
Recover Genes
Subclone into an expression vector
Assay Survival
Tx Menadione
Neurons Fibroblasts
SequenceBioinformatics
Tx MenadionePCR
Functional Cloning of NeuroProtective Genes
Control Menadione
C139SC2
Don
g Li
ang
Differential Analysis of Primary Library
Expression (DAzLE)
Proc. Natl. Acad. Sci. U.S.A., 101: 647-652, 2004Proc. Natl. Acad. Sci. U.S.A., 101:2145-2150, 2004
Screening for Survival Genes/Proteins
Functional Cloning
DAzLE
Strengths
WeaknessCannot distinguish endogenous vs. preconditioning survival genesCannot conduct in excitable cells (neurons)Genes must be FL-ORF
Genes are functionalKnow that genes mediate survivalWill identify pathways of survival in non-neuronal cells
Genes are induced by PC stressWill identify gene fragments, full length can be cloned laterCan be done in excitable cells (neurons)Need additional experimental design to identify survival genesRequires a lot of sequencingAdditional cloning required
Protection Against Toxic Menadione
Neu
ropr
otec
tion
OGD
NMDA
DAzLE PC Gene Set inMaximal Electroconvulsive Shock (MECS)
MECS ± MK801 12hr-24hr after MECS
2424+MK801+MK801
1212 24241212
DAzLE: Functional Categories of NMDA-induced Genes
NMDA Activated GenesStress Genes
PreconditioningGenes
How to find PC Genes?
NMDA-induced PreconditioningNMDA-induced Preconditioning
NMDANMDA
NMDA-R
NOS
p21 Ras
Raf
Mek
Erk
New Transcription / Translation
Preconditioning Mediated Neuroprotection
N-nitro-argininenNOS null vs WT
PD98059
50 50 M NMDA, 5 minM NMDA, 5 min
1hr-3hr-6hr-12hr-24hr1hr-3hr-6hr-12hr-24hrMEK/NO inhibitorMEK/NO inhibitor
NMDA-inducedGenes
658 Genes
MEKPathway
246 Genes
129Genes
NOPathway
218 Genes
31Genes
108Genes
12Genes
Venn Diagram of Differentially Regulated GenesVenn Diagram of Differentially Regulated Genes
Antisense Knock-down of Selected Genes Antisense Knock-down of Selected Genes Abolishes PreconditioningAbolishes Preconditioning
Cel
l D
eath
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
Precond. 435 609 707 725 932 1012 Excito
****
** ****
** P < 0.001
Sense Antisense
(red: dead neurons)
Iduna: Norse Goddess of Immortality and Healing
Iduna protects cortical neurons from NMDA excitotoxicity
Iduna sequence
***
***PAR Binding
Localization of Iduna1˚ Cortical Cultures
Suk Jin Hong
Regional Expression of IdunaN
orth
ern
Blo
tW
este
rn B
lot
Iduna is Induced by PreconditioningCortical Cultures
Mouse Cortex
Suk Jin Hong JHMISunghee Cho & Constantino IadecolaWeill College of Medicine at Cornell
CA1 Control CA1 BCCAO 24hbilateral common carotid occlusion
CA1 Control CA1/CA2 BCCAO 24h
Cortex Outer Layer BCCAO 24h
Cortex Inner Layer BCCAO 24h
Cortex Outer Layer Naïve Control
Cortex Inner Layer Naïve Control
Cortex BCCAO 24hCortex Naïve Control
Mutant Constructs
32P-pADPr Overlay Blot: -GFP
56
36
29
101
GFPId
una-EGFP
GFPId
una-EGFP
contro
l
Iduna is a PAR binding protein
RING-finger WWEYR
Iduna
PAR
0
20
40
60
80
100
120
Control WWE YR-AA
% C
on
trol C
ell
Death
Iduna
*
*p < 0.001, Student’s t-test
500 µM NMDA
0
20
40
60
80
100
120
Control WWE YR-AA
% C
on
trol C
ell
Death
Iduna
*
*p < 0.001, Student’s t-test
MNNG
Mutation of the PAR Binding Site Prevents Neuroprotection by Iduna
RF
*
Thoughts for the Future……There are novel survival proteins and
pathways in the brain that need to be uncovered and understood.These survival proteins/pathways have tremendous promise as future therapeutic targets.With appropriate experimental design, gene screening is a powerful tool to reveal new survival pathways.Hopefully, understanding novel survival pathways may illuminate novel death pathways in the nervous system.
AcknowledgmentsAcknowledgmentsTed M. DawsonTed M. DawsonSuk Jin HongSuk Jin HongXiujing GuXiujing GuHuiwu LiHuiwu LiDong LiangDong LiangCheng DaiCheng DaiMirella Gonzalez-Mirella Gonzalez-ZuluetaZuluetaHye-Young YunHye-Young YunSika ZhengSika Zheng
NIH-NINDS, NIH-NIDAAHA, McKnight Foundation, Abramson Foundation
Kevin BeckerKevin BeckerLaura J. KlesseLaura J. KlesseRobert G. KalbRobert G. KalbLuis F. ParadaLuis F. ParadaGuy PoirerGuy PoirerJeff HainceJeff HainceConstantino Constantino
IadecolaIadecolaSunghee ChoSunghee Cho